Cell culture system

ABSTRACT

Disclosed is a method for the culture of higher eukaryotic cells which are dependent for survival on an exogenous factor. The method involves co-culturing the factor-dependent cells with an immortalized eukaryotic cell that has been engineered to secrete the requisite factor. 
     Also disclosed is a cell line of non-stromal cell origin which secretes interleukin-7.

This is a divisional of application Ser. No. 08/342,643, filed Nov. 21,1994, now U.S. Pat. No. 5,459,058 which is a continuation of applicationSer. No. 08/119,315, filed Sep. 9, 1993, now abandoned, which is acontinuation of application Ser. No. 07/939,976, filed Sep. 4, 1992, nowabandoned, which is a continuation of application Ser. No. 07/676,816,filed Mar. 28, 1991, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to in vitro cell culture systems.

Given the appropriate conditions, many types of mammalian cells may bepropagated in vitro. Typically, such conditions include achemically-defined growth medium containing essential amino acids (e.g.,cysteine, glutamine, and tyrosine), vitamins (e.g., biotin, choline,folic acid, thiamine), salts (e.g., NaCl, KCl, CaCl₂), glucose, and, inmany cases, serum. The role of serum is not completely understood,however it is purported to provide requisite trace materials, e.g.,protein growth factors. In addition to a defined culture medium, manycultured mammalian cells require a solid surface on which to grow anddivide. This requirement is most often provided by a glass or plastictissue-culture dish or flask. In the absence of such a requirement,cells may be grown in suspension, e.g., in a test tube or tissue-culturebottle.

Two types of cultured mammalian cells are in widespread experimental andindustrial use: "primary cells" and "immortalized cells". Primary cellsare prepared directly from the normal tissues of an organism, e.g., fromskin, bone marrow, or whole embryos. These cells grow well when firstplaced under culture conditions, typically surviving for weeks or monthsand dividing for about 50-100 generations. At that time, cells reach astage, termed "crisis"; they grow and divide slowly, and soon,thereafter, cease to grow and divide altogether.

Immortalized cells are characterized by indefinite growth in vitro.Typically, immortalized cell cultures are derived either from a tumorsample explanted directly from an organism or from a primary cellvariant which has undergone a change which promotes indefinite growth.

Hematopoietic cells may be propagated as primary cell cultures in vitro.Growth factors are provided, either by exogenous addition of the factorsto the culture medium or by secretion of the factors by "feeder cells".Such feeder cells are generally stromal cells (i.e., a poorly definedmix of primary cells derived from bone marrow). A description ofhematopoietic cell culture involving a stromal cell feeder layer isdescribed by Whitlock et al. (J. Imm. Methods 67:353, 1984) and Whitlockand Witte (Meth. Enzymol 150:275, 1987); using their method, continuouscultures of early B-lymphocytes are established on an adherent feederlayer of dissociated mouse bone marrow cells. There is some support forthe idea that hematopoietic cell growth also requires cell-cell contact.For example, Kincade et al. (Curr. Topics in Micro. & Imm. 135:1, 1987)report the dependence of cultured lymphoid cells on substances producedby, and/or on physical association with, an adherent stromal cell layer.

SUMMARY OF THE INVENTION

In general, the invention features a method for culturing a highereukaryotic cell which is dependent for survival upon an exogenousfactor. The method involves co-culturing the factor-dependent cell withan immortalized higher eukaryotic cell that has been engineered tosecrete the requisite exogenous factor.

In preferred embodiments, the cell dependent for survival upon anexogenous factor is further dependent for survival on cell-cell contact;is a non-adherent cell; is a mammalian cell, preferably, a human cell;and is a ematopoietic cell, preferably, a B-lineage lymphocyte, aT-lineage lymphocyte, or a thymocyte; the immortalized higher eukaryoticcell is an adherent cell; is a mammalian cell, preferably, a human cell;and is a fibroblast cell, preferably, an NIH3T3 cell; the exogenousfactor is interleukin-7; the interleukin-7 is expressed from a stablytransfected gene in the immortalized higher eukaryotic cell; the stablytransfected gene includes an enhancer; the interleukin-7 is encoded byplasmid pBAIL-7; and the culturing is long-term.

In another preferred embodiment, the method further involves contactingthe immortalized higher eukaryotic cell with a growth-inhibitory amountof mitomycin-C just prior to co-culturing with the higher eukaryoticcell which is dependent for survival upon an exogenous factor.

In a related aspect, the invention features a cell line of non-stromalcell origin that secretes interleukin-7. In preferred embodiments, thecell line harbors a transgene that directs expression of interleukin-7;the transgene comprises an enhancer, preferably, an actin enhancer; thecell line is a mammalian cell line, preferably, a fibroblast cell line,more preferably, an NIH3T3 cell line, and, most preferably, NAIL-7.

By "higher eukaryotic cell" is meant a cell which contains a nucleus andis derived from a multicellular organism. The term, as used herein, doesnot include a yeast cell. By "dependent for survival upon" is meant torequire for survival and/or growth or proliferation. By "immortalized"ismeant able to grow (i.e., produce progeny cells) indefinitely in cellculture. By "non-stromal cell origin" is meant not deriving from a bonemarrow explant. By "secrete" is meant to export a substance, e.g., afactor from the inside to the outside of a cell. The term includesexport by either active or passive mechanisms. By "factor" is meant anysubstance which a cell requires to survive and/or grow and/orproliferate and which can be produced and exported by another cell. Suchfactors include, without limitation, growth factors (e.g., interleukins,insulin, transferrin, hydrocortisone, fibroblast growth factor, nervegrowth factor, epidermal growth factor), amino acids, and vitamins. By"cell-cell contact" is meant a physical association between two cells.By "adherent" is meant capable of maintaining contact with a solidsupport for a substantial period of time; the solid support is,preferably, a cell culture dish or flask and the substantial period oftime is, preferably, all or most of the generation time of the culturedcell. By "stably transfected gene" is meant a piece of DNA encoding aprotein which is inserted by artifice into a cell and becomes heritablytransmitted to progeny cells. Such a stably transfected gene may bepartly or entirely heterologous to the host cell and may be insertedinto the host cell genome at the same or at a different location (e.g.,a modified gene which is homologously recombined into the host genomereplacing the endogenous genomic sequence); alternatively, such a stablytransfected gene may be a gene homologous to a natural gene of the hostcell, but which is inserted into the host cell's genome at a locationwhich differs from that of the natural gene. By an "enhancer" is meant acis-acting DNA sequence capable of increasing transcription from apromoter that is located either upstream or downstream of the enhancerregion. Such enhancer DNA sequences are well known to those skilled inthe art of eukaryotic gene expression. By "B-lineage lymphocytes" ismeant a hematopoietic cell which matures into an immunoglobin-producingcell. By "thymocyte" is meant a cell which resides during part of its invivo existence in the thymus and which is a precursor to a "T-lineagelymphocyte". By "hematopoietic cell" is meant a blood cell whetherdevelopmentally mature or immature, or a precursor of a blood cell. By"long-term" culture is meant culture which extends for a continuousperiod of time which is generally greater than one month.

This invention is based on the discovery that NIH 3T3 cells (i.e.,immortalized, higher eukaryotic cells) that have been engineered toproduce IL-7 can support the long-term growth and proliferation offactor-dependent hematopoietic cells, in particular, B-lineagelymphocytes. Such feeder layers stimulate rapid B-lineage lymphocyteproliferation and permit a high final B-lymphocyte cell density.

The invention provides a convenient alternative to the culture offactor-dependent cells on primary feeder layers. For example, themethods of the invention do not require the sacrifice of animals (e.g.,mice) nor do they involve an extended period of time for establishmentof a feeder layer (e.g., 2-4 weeks for establishment of a stromal cellfeeder layer). In addition, the feeder layers of the invention arecomposed of a clonal population of cells. This facilitates a predictableand reproducible method of cell culture through the provision of definedgrowth conditions. Use of a clonal feeder layer also circumventsproblems of "contamination" by undesirable cells (e.g., those present ina stromal cell population) which grow very rapidly and compete with thefactor-dependent cells for nutrient sources and/or cell-cell contact. Inthe worst case, contaminating cells may actually destroy thefactor-dependent cells through secretion of toxins or by phagocytosis.

Other features and advantages of the invention will be apparent from thedescription of the preferred embodiments, and from the claims.

DETAILED DESCRIPTION OF DRAWINGS

The drawings will first briefly be described.

FIG. 1 is a diagram of the IL-7 expression vector, pBAIL7.

FIG. 2 is a graph showing lymphocyte proliferation on three differentadherent mammalian cell lines.

FIG. 3 is a representation of a series of micrographs showingB-lymphocytes cultured on an adherent layer of (A) primary bonemarrow-derived feeder cells, (B) NAIL-7 feeder cells, and (C) NIH 3T3feeder cells.

There now follows one example of an immortalized mammalian cell line(i.e., the NAIL-7 cell line) which has been engineered to produce andsecrete a factor (i.e., IL-7) and a description of its use as a feederlayer for the culture of hematopoietic cells (i.e., B-lineage cells)which are dependent for their survival upon both the factor andcell-cell contact. This example is presented for the purpose ofillustrating, not limiting, the invention.

The NAIL-7 Cell Line.

The pBAIL-7 expression plasmid and the NAIL-7 cell line were constructedas follows.

A cDNA library was first prepared. mRNA was extracted from the spleen ofa Swiss mouse by the guanidinium isothiocyanate method of Chirgwin(Biochemistry 18:5294, 1976), and cDNA produced from this RNA using thecDNA synthesis kit of Boehringer-Mannheim (Indianapolis, IND.) and therecommendations of the manufacturer. Oligodeoxynucleotide PCR primerswere designed based on the published IL-7 sequence of Namen et al.(Nature 333:571, 1988) and generated by an Applied Biosystems 380A DNASynthesizer (Applied Biosystems, Foster City, Calif.). The sequences ofthe primers corresponded to nucleotides 525-551 of the IL-7 positivesense strand and 1158-1184 of the IL-7 negative sense strand. Inaddition, four nucleotides, GGTC, were included at the 5' end of eachprimer to create a SalI restriction enzyme recognition site in the PCRproduct. 0.1×10⁹ moles of each PCR primer were mixed with 4μg of spleencDNA, and an IL-7 cDNA molecule was amplified by the polymerase chainreaction (PCR) procedure of the Cetus Corporation (Perkin-Elmer CetusGene Amp Kit, Norwalk, Conn.). PCR-amplified reaction products weretreated with polynucleotide kinase to add 5' terminal phosphate groupsand the fragments were ligated to SmaI-digested, pBluescript KS(Stratagene, La Jolla, Calif.). E. coli strain DH5α was transformed withthe ligation mixture and ampicillin-resistant cells (i.e., those cellsharboring a recombinant plasmid) were selected and propagated in cultureby standard techniques (see, e.g., Ausubel et al., Current Protocols inMolecular Biology, Wiley Publishing, New York, N.Y., 1987). Plasmid DNAwas prepared by standard techniques (Ausubel et al., supra), and thesequences of the cDNA inserts were determined by the method of Sanger(Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463, 1977). ClonepIL-7.16 was shown to contain a full-length IL-7 coding sequence and wasused for all subsequent subcloning procedures.

To direct expression of IL-7 in NIH3T3 cells, the IL-7 cDNA was inserteddownstream of (i.e, under the transcriptional control of) the humanβ-actin enhancer and promoter, transcriptional control elements whichhave been shown to be highly active in NIH3T3 cells (Gunning et al.,Proc. Natl. Acad. Sci. USA 84:4831, 1987). The expression plasmid wasconstructed as follows. pIL7.16 was digested with SalI, and a 668 basepair fragment containing the IL-7 CDNA was isolated by agarose gelelectrophoresis (Ausubel et al., supra). This fragment was ligated toSalI-digested pHBApr-1 (Gunning et al., Proc. Natl. Acad. Sci. USA84:4831, 1987) and used to transform E. coli DH5βhost cells as describedabove. Plasmid DNA was prepared from several independent colonies andwas screened by restriction digestion analysis using the published IL-7and pHBApr-1 sequences described above. A recombinant plasmid with theIL-7 cDNA in the appropriate orientation was selected and termed pBAIL-7(β-Actin/IL-7).

pBAIL-7 was introduced into cultured mammalian NIH3T3 cells as follows.20 μg of ScaI-digested pBAIL-7 and 1 μg of EcoRI-digested pSV7neo(Murphy et al., Proc. Natl. Acad. Sci. USA 83:2939, 1986) were mixedwith 10⁷ NIH3T3 fibroblasts (ATCC Accession Number CRL-6442) in 0.5 mlof phosphate buffered saline. pSV7neo confers resistance to G418, andcells transfected with this plasmid may be dominantly selected (Ausubelet al., supra). Cells were electroporated by standard techniques using aBioRad Gene Pulser machine (0.25 Kv, 960 uF) (BioRad, Hercules, Calif.;Ausubel et al., supra), incubated for two days in nonselective medium(i.e., Dulbecco's Modified Eagle Medium, or DMEM, supplemented with 2 mMglutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% bovinecalf serum; GIBCO, Grand Island, N.Y.), and then incubated for 11 daysin selective medium (i.e., DMEM containing 400 μg/ml G418; Geneticin;Life Technologies Inc., Grand Island, N.Y.). Twelve G418-resistantclones were selected and transferred to individual plates with acapillary tube. Clones were propagated for several generations inselective medium, aliquots of cells were then propagated innon-selective medium, and their culture supernatants were assayed forthe production of IL-7 by the method of Namen et al. (J. Exp Med.164:988-1002, 1988). In addition, RNA was prepared from samples of theclones by the method of Chirgwin (Biochemistry 18:5294, 1976) andassayed for the presence of IL-7 MRNA by RNase protection (by the methodof Krieg et al., Nucl. Acids Res. 12:7035-56, 1984) using anIL-7-specific probe including nucleotides 525 to 656 of the anti-sensestrand of pIL7.16. One cell line shown to express IL-7 MRNA and secreteactive IL-7 was chosen for experiments described herein. This cell linewas termed NAIL-7 (NIH3T3/β-Actin/IL-7).

Lymphocyte Cultures

A monolayer of NAIL-7 cells was used to support the growth of B-lineagelymphocytes and the efficacy of such a NAIL-7 feeder layer was comparedto that of a parental NIH3T3 cell layer and a primary stromal celllayer.

Primary stromal cell feeder layers were prepared by the method ofWhitlock et al. (J. Imm. Meth. 67:353-369, 1984). NAIL-7 and NIH3T3 cellfeeder layers were prepared by treating confluent cultures of each celltype with mitomycin-C (10 μg/ml for 2-4 hr., Sigma Chemical Co., St.Louis, Mo.) to block cell division. Following treatment, cells werereplated at 5×10⁴ cells/cm².

Feeder-dependent B-lineage lymphocytes were prepared from BALB/c mice bythe method of Whitlock et al., (J. Imm. Meth. 67:353, 1984) and wereplated at 10⁵ cells/ml in dishes containing monolayers of either stromalcells, mitomycin-C-treated NIH3T3 cells, or mitomycin-C-treated NAIL-7cells (each prepared as described above). At 3-4 day intervals, thecultures were alternately supplemented with a half-volume of selectivemedium, or resuspended vigorously, aspirated, and replenished with onevolume of selective medium. The cells in the aspirates were counted bystandard techniques.

Results presented in FIG. 2 show the number of B-lymphocytes in culturesgrown on either NAIL-7 cells (▴), NIH3T3 cells (▪), or bone marrowstromal cells (). FIG. 3 shows feeder-dependent lymphocytes grown for10 days on either NAIL-7 cells (middle panel, B) or NIH3T3 cells (rightpanel, C). FIG. 3 also shows a six week culture of bone marrow stromalcells (left panel, A). FIGS. 2 and 3 indicate that feeder-dependentlymphocytes cultured on primary bone marrow stromal cells or on NAIL-7cells proliferated vigorously while the same lymphocytes plated onNIH3T3 cells grew very poorly, if at all. FIG. 2 further shows thatB-lymphocytes grow rapidly and to a higher density on NAIL-7 cells thanon a stromal cell feeder layer or on an NIH3T3 cell layer.

Certain aspects of this method may be altered without destroying theefficacy of the culture system. For example, although it is preferableto routinely transfer aliquots of the cultured B-lymphocytes to platescontaining freshly mitomycin C-treated NAIL-7 cell layers (e.g., once aweek to once a month), a continuous culture of B-lymphocytes may besuccessfully propagated on a single layer of treated NAIL-7 cells formany months. To date, B-lymphocytes have been cultured for up to eightcontinuous months on the same layer of mitomycin-C treated NAIL-7 cells.

B-lymphocytes have also been propagated on a NAIL-7 cell line which wasnot treated with mitomycin-C. Culture was carried out as described above(i.e., using equivalent medium lacking mitomycin-C) except that theNAIL-7 feeder layer was plated at a very low initial density (e.g., at1/10 confluence or approximately 10⁴ cells or less/10 cm² plate). Thelength of continuous culture time is limited by the rapid growth of theuntreated NIH3T3 feeder layer and the resultant exhaustion of culturenutrients.

Examples of Other Culture Systems

The method of the invention can be used to culture factor-dependentcells remote from B-lineage lymphocytes and can utilize feeder celllayers other than IL-7-producing NIH3T3 cells. The following examplesillustrate that such feeder cells and such factor-dependent cells may beeither adherent or non-adherent. These examples are designed to provideguidance and should not be construed as limiting.

Culture of adherent factor-deDendent cells with non-adherent feedercells

In a first example, adherent factor-dependent cells are propagated withnon-adherent feeder cells The factor-dependent cells are plated on asolid support, e.g., on a tissue culture dish, under conditions and fora period of time which allow adherence to the solid support. Feedercells are then suspended in the culture medium, where they secretefactors which promote adherent cell growth. Harvesting thefactor-dependent cells involves removal of the feeder cells byaspiration of the culture supernatant followed, where necessary, by oneor more washing steps with, e.g., phosphate buffered saline. Thefactor-dependent cells are then collected following their release fromthe solid support, e.g., by brief treatment with trypsin (by standardmethods; see, e.g., Ausubel et al., supra).

In particular, this method of this first example may be used for thepropagation of adherent factor-dependent endothelial cells withengineered non-adherent hematopoietic cells, e.g., lymphocytes. Theendothelial cells and hematopoietic cells are cultured by standardtechniques (see, e.g., Andus et al., Pharm. Res. 7:435, 1990; andmethods described herein, respectively). The hematopoietic cells areengineered to produce a factor which stimulates endothelial cell growth,such as fibroblast growth factor, transforming growth factor-α,transforming growth factor-β, or the c-kit ligand (see, e.g., Folkman etal., Science 235:442, 1987; Ingber et al., J. Cell Biol. 109:317, 1989).Following propagation, the endothelial cells are harvested as decribedabove.

Culture of adherent factor-dependent cells with adherent feeder cells

In a second example, both the factor-dependent cells and the feedercells are adherent. In this case, cells are plated simultaneously andallowed to adhere to a solid support (preferably, a tissue culture dish,as generally described above). The factor-dependent cells are stimulatedto divide by growth factors produced by the adherent feeder cells.Following propagation, the factor-dependent cells and the feeder cellsare simultaneously harvested by brief treatment with trypsin (asdescribed above). Factor-dependent cells are isolated from the mixedpopulation by physical methods designed to differentiate between the twocell types, e.g., elutriation or density gradient centrifugation(Beckman Publications DS 534; Beckman Instruments, Columbia, Md.;Ausubel et al., supra; respectively). The factor-dependent cells mayalso be isolated by immunological methods, such as binding to celltype-specific monoclonal antibodies attached to plastic plates ormagnetic beads (Wysocki and Sato, Proc. Natl. Acad. Sci. USA 75:2844,1978; Dynal, Inc, Great Neck, N.Y.), or to fluorescent molecules forfluorescence activated cell sorting (Jovin et al., Trends Biochem. Sci.5:214, 1980; Herzenberg et al., Sci. Am. 234:108, 1976; OrthoDiagnostics Systems, Inc., Westwood, Mass.).

In specific examples, this method is useful for the culture offactor-dependent endothelial cells or factor-dependent nerve cells on anengineered NIH3T3 feeder layer. In the first case, the feeder layercells are engineered to produce endothelial cell growth factors such asfibroblast growth factor, transforming growth factor-α, transforminggrowth factor-β, or kit ligand (see, e.g., Folkman et al., 1987, supra;Ingber et al., 1989, supra). In the second case, the immortalizedadherent cells are engineered to produce a nerve cell growth factor suchas nerve growth factor (NGF) (see, e.g., Bienenstock et al., Int. Arch.Allergy Appl. Immunol. 87:238, 1987; Carbonetto et al., J. Physiol.(Paris) 82:258, 1987). Cells are grown using standard methods ofendothelial cell or nerve cell culture (see, e.g., Audus et al., Pharm.Res. 7:435, 1990; and Lander, Mol. Neurobiol. 1:213, 1987; Bunge et al.,Prog. Brain Res. 78:321, 1988; Azmitia et al., Neurobiol of Aging 9:743,1988, respectively). The factor-dependent cells are separated byphysical or immunological methods as described above.

Culture of non-adherent factor-dependent cells with nonadherent feedercells

Finally, both the factor-dependent cells and the feeder cells may benon-adherent. In this case, factor-dependent cells and feeder cells arecultured as a mixed population in solution (e.g., in a test tube orculture bottle). Once propagated, the factor-dependent cells areharvested by physical or immunological separation from the feeder cells,using, for example, the methods described above. Alternatively,factor-dependent cells not requiring cell-cell contact may be grown insolution with feeder cells, under conditions where the factor-dependentcells remain separated from the feeder cells by some physical barrier,e.g., a semi-permeable membrane. Such a physical barrier must preventcells from mixing but must allow factor(s) to pass from the feeder cellsto the factor-dependent cells. Preferably, the physical barrier used forthis type of culturing is provided by a Millicell-CM (Millipore,Bedford, Mass.).

In a specific example, this method is used for the culture offactor-dependent B-lymphocytes with engineered T-lymphocyte feedercells. Cells are grown using standard methods of lymphocyte cell culture(see, e.g., methods described herein). The feeder cells are engineeredto produce a B-lymphocyte growth factor, such as IL-7 (as describedherein). If the factor-dependent and feeder cells are cultured togetherin suspension, the factor-dependent cells are separated by physical orimmunological methods as described above. Alternatively, if thefactor-dependent cells and feeder cells are cultured on opposite sidesof a semi-permeable membrane, the need for later separation iscircumvented, and the factor-dependent cells may be harvested directlyfrom suspension.

For all of the above examples, the choice of culture conditions (e.g.,choice of growth medium) will vary slightly according to the types offeeder and factor-dependent cells involved; appropriate conditions arewell known by those skilled in the art. Where necessary, cultureconditions are adjusted to accommodate the growth condition requirements(e.g., media requirements) of both the feeder cells and thefactor-dependent cells, the modifications are generally minor and shouldnot be an impediment to the success of the culture method.

Other Embodiments

Mitomycin-C treatment is not necessary to the success of the instantculture method. Mitomycin-C treatment facilitates the use of feederlayers capable of rapid proliferation; however, as described above,untreated feeder layers have been used successfully (even veryrapidly-dividing NIH3T3 feeder layers). Moreover, since mitomycin-Ctreatment simply slows feeder layer proliferation, it may be eliminatedwhen the culture system makes use of a slowly-dividing feeder layer.

The method of the invention may be used for the propagation of humancells, for example, B- or T-lineage lymphocytes. Because the culturesystem provides the opportunity to define the growth conditions,particular growth factors may be provided by the feeder layer whichstimulate maturation of, e.g., blood cells from progenitor cells.Alternatively, omission of a growth factor which triggers celldifferentiation (e.g., one which is normally secreted by primary celllayers, such as stromal cells) allows the large-scale propagation ofcell precursors, e.g., stem cells. In one particularly usefulapplication, human cells are isolated from a patient, e.g., stem cellsor blood cells from an immunodeficient or immunocompromised patient. Thecells are propagated using the in vitro method of the instant invention,harvested, and re-introduced (e.g., intravenously) into the patient tobolster or restore immune function.

We claim:
 1. A method for culturing an animal cell which is dependentfor survival upon an exogenous factor, said method comprisingco-culturing said cell with an immortalized animal cell that expressesand secretes said factor.
 2. The method of claim 1, wherein said celldependent for survival upon an exogenous factor is further dependent forsurvival on cell-cell contact.
 3. The method of claim 1, wherein saidcell dependent for survival upon an exogenous factor is a non-adherentcell.
 4. The method of claim 1, wherein said cell dependent for survivalupon an exogenous factor is a mammalian cell.
 5. The method of claim 4,wherein said mammalian cell is a human cell.
 6. The method of claim 1,wherein said cell dependent for survival upon an exogenous factor is ahematopoietic cell.
 7. The method of claim 6, wherein said hematopoieticcell is a B-lineage lymphocyte.
 8. The method of claim 6, wherein saidhematopoietic cell is a T-lineage lymphocyte.
 9. The method of claim 6,wherein said hematopoietic cell is a thymocyte.
 10. The method of claim1, wherein said immortalized animal cell is an adherent cell.
 11. Themethod of claim 1, wherein said immortalized animal cell is a mammaliancell.
 12. The method of claim 11, wherein said mammalian cell is a humancell.
 13. The method of claim 1, wherein said immortalized animal cellis a fibroblast cell.
 14. The method of claim 13, wherein saidfibroblast cell is an NIH3T3 cell
 15. The method of claim 1, whereinsaid culturing is long-term.
 16. The method of claim 1, furthercomprising contacting said immortalized animal cell with agrowth-inhibitory amount of mitomycin-C just prior to co-culturing withsaid animal cell which is dependent for survival upon an exogenousfactor.
 17. The method of claim 1, wherein said cell dependent forsurvival upon an exogenous factor is a lymphoid cell.
 18. A method forculturing a lymphoid cell which is dependent for survival upon aninterleukin, said method comprising co-culturing said cell with animmortalized animal cell that expresses and secretes said interleukin.19. The method of claim 18, wherein said cell dependent for survivalupon an exogenous factor is a hematopoietic cell.
 20. The method ofclaim 18, wherein said cell dependent for survival upon an exogenousfactor is a lymphoid cell.